SITC 2016, ABSTRACT: Intralesional injection with Rose Bengal and systemic chemotherapy induces anti-tumor immunity in a murine model of pancreatic cancer

Updated below: 11/8/16 {thrice}.

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Intralesional injection with Rose Bengal and systemic chemotherapy induces anti-tumor immunity in a murine model of pancreatic cancer

Shari Pilon-Thomas, Amy Weber, Jennifer Morse, Krithika Kodumudi, Hao Liu, John Mullinax, Amod A Sarnaik H. Lee Moffitt Cancer Center, Tampa, FL, USA

Journal for ImmunoTherapy of Cancer 2016, 4(Suppl 1):P256

Background
Rose Bengal is a xanthene dye that has been utilized for liver function studies and is currently used topically in ophthalmology. Intralesional (IL) Rose Bengal (PV-10) has been shown in murine models and melanoma clinical trials to induce regression of treated melanoma lesions and uninjected bystander lesions. This study was undertaken to measure whether IL PV-10 can induce systemic anti-tumor effects alone or in combination with gemcitabine (Gem) therapy in a murine model of pancreatic cancer.

Methods
C57BL/6 mice received Panc02 pancreatic tumor cells subcutaneously (SC) on one flank to establish a single tumor. On day 7, tumor was treated with IL PV-10. Control mice received IL phosphate
buffered saline (PBS). Tumor growth was measured. Splenic T cells were collected and co-cultured with Panc02 or irrelevant B16 cells. Supernatants were collected to measure Panc02-specific T cell responses by IFN-gamma ELISA. To measure the effect of IL PV-10 on the growth of an untreated, bystander tumor, mice received Panc02 cells in bilateral flanks. The resulting right tumor was injected IL with PV-10 or PBS. Tumor sizes were measured for both the right (treated) and left (untreated/bystander) tumors. To determine the efficacy of combination therapy with IL PV-10 and systemic Gem, mice bearing a single or bilateral Panc02 tumors were treated with PV-10 alone or in combination with Gem. Mice received 60 mg/kg Gem intraperitoneally (IP) twice per week.

Results
C57BL/6 mice bearing Panc02 tumors treated with IL PV-10 had significantly smaller tumors than mice treated with PBS (p < 0.001). A significant increase in the IFN-gamma production in response to Panc02 was measured in the splenocytes of mice treated with PV-10 as compared to mice treated with PBS (p < 0.05). Mice with bilateral tumors had a significant regression of tumors injected IL with PV- 10 and there was a reduction in the untreated (bystander) flank Panc02 tumor (p < 0.01). Gem therapy in combination with IL PV-10 injection led to enhanced tumor regression (p < 0.05) compared to IL PV-10 or Gem alone in both a single tumor model and a bilateral tumor model.

Conclusions
Regression of untreated pancreatic tumors by IL injection of PV-10 in concomitant tumor supports the induction of a systemic anti-tumor response. Addition of Gem chemotherapy enhances the effects of IL PV-10 therapy. Given that patients with metastatic pancreatic cancer have a dismal prognosis, combination therapy of IL PV-10 combined with Gem may benefit patients with metastatic pancreatic cancer.

Updated (11/8/16).1: My underlined emphasis above. Note PV-10 use in the above murine model work as a monotherapy, and in combination with systemic chemotherapy.

Updated (11/8/16).2: H/t @bradpalm1:

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"Gemcitabine reduces MDSCs, tregs and TGFβ-1 while restoring the teff/treg ratio in patients with pancreatic cancer," Eriksson et al., Journal of Translational Medicine 2016 14:282

"Conclusions 

Gemcitabine regulates the immune system in patients with pancreatic cancer including MDSCs, Tregs and molecules such as TGFβ-1 but does not hamper the ability of effector lymphocytes to expand to stimuli. Hence, it may be of high interest to use gemcitabine as a conditioning strategy together with immunotherapy."

Updated (11/8/16).3: Unlike some, I do not read too much into Moffitt's abstract with respect to the additivity or synergism of PV-10 and gemcitabine (systemic chemotherapy) based on the cancer center's murine model work (i.e., p values of PV-10 alone, and in combination with chemo). And, I'd like to see the poster if Provectus facilitates its release (e.g., the company did not facilitate the release of Moffitt's AACR 2016 poster, which I believe may lead to a peer-reviewed publication).

I believe the point of this mousie work, which of course is beyond cell line (in vitro) work but behind clinical studies, is to demonstrate in principle that (a) intralesional (IL) PV-10 could be used to treat pancreatic cancer (i.e., tumor type, leading to a suitable cancer indication) via (i) ablation/destruction of an injected tumor and (ii) the subsequent triggering of the immune response to reduce or destroy an untreated one, and (b) IL PV-10 plus chemo sees enhanced untreated tumor reduction or destruction.

These principles of (x) ablation/destruction by injection and (y) immunologic signalling (immune system harnessing) already have been shown preclinically by Moffitt for PV-10 as a monotherapy in melanoma (also clinically) and breast cancer (AACR 2013), and in combination with checkpoint inhibition for melanoma (SITC 2014).

So, this continues Rose Bengal, PV-10 and Provectus' theme of (A) agnosticism (ablation, immunologic signalling), (B) synergism (in that one therapy enhances another; "induce and boost"), (C) orthogonality (although this would be better shown in clinical work to emphasize no greater toxicity, if not less to far less), and...

(D) PV-10 is an immunotherapy. See January 19, 2016 blog post PV-10 is an immunotherapy.

SITC 2016: Intralesional injection with Rose Bengal and systemic chemotherapy induces anti-tumor immunity in a murine model of pancreatic cancer

Updated below: 9/26/16 {twice}, 9/28/16 and 9/30/16.

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Updated (9/26/16): According to the Pancreatic Cancer Action Network, there are three chemotherapy drugs approved by the United States Food and Drug Administration (FDA) for the treatment of pancreatic cancer: albumin-bound paclitaxel (ABRAXANE®), gemcitabine (Gemzar®) and fluorouracil (5-FU).

"Gemcitabine (Gemzar®) was approved in 1996 for the treatment of unresectable pancreatic cancer. Studies have also shown that there is a benefit to using gemcitabine after surgery for pancreatic cancer. Prior to gemcitabine, fluorouracil (5-FU) was used as the standard treatment for unresectable pancreatic cancer. Both of these drugs are still used today. 

Most recently, in September 2013, albumin-bound paclitaxel (ABRAXANE®) was approved to be used in combination with gemcitabine (Gemzar®) as first-line treatment for metastatic pancreatic adenocarcinoma, the most common type of pancreatic cancer. 

In addition to the three FDA-approved drugs, FOLFIRINOX, a combination of three chemotherapy drugs (5-FU/leucovorin, irinotecan, and oxaliplatin) is commonly used in the treatment of metastatic pancreatic adenocarcinoma. In 2010, a Phase III clinical trial showed positive results for patients treated with FOLFIRINOX. Due to the results of this study, FOLFIRINOX is also considered a standard treatment option for patients with metastatic pancreatic cancer. However, patients treated with FOLFIRINOX may experience more severe side effects than those treated with gemcitabine alone, so this combination is usually given to patients who are healthy enough to tolerate the potential side effects."

Abraxane is made by Celgene. Gemzar is made by Eli Lilly. Provectus previously showed the combination of 5-FU and PV-10 for hepatocellular carcinoma in murine model work (SITC 2012).

Updated (9/26/16): I believe the systemic chemotherapy used in Moffitt's work is gemcitabine (Gemzar®), which by now is produced by generics manufacturers.

Some recent information on the competitive landscape for pancreatic cancer:

• "Eli Lilly's chemotherapy drug Gemzar (gemcitabine) is widely considered the standard of care for pancreatic cancer. Generic gemcitabine is available from numerous companies" {January 2014 source},

• "In the past, companies have combined various targeted treatments with gemcitabine in an attempt to increase their survival benefit. These efforts include Bristol-Myers Squibb/Eli Lilly's Erbitux, Roche's Avastin, Pfizer's Inlyta, and Amgen/Bayer's Nexavar, among others. Unfortunately, none of these drugs has shown a significant survival benefit when combined with gemcitabine" {above source},

• "Evolving Paradigms in Pancreatic Cancer: Frontline Treatment Regimens:"

• "Frontline therapies for patients with metastatic pancreatic cancer have advanced in the past 5 years, with the FDA approval of nab-paclitaxel (Abraxane) plus gemcitabine (Gemzar). These advancements branch off the established treatment paradigm of gemcitabine monotherapy, which showed a significant extension in overall survival (OS) compared with 5-FU alone in a phase III study."

• "Pancreatic cancer is a particularly tough cancer to treat and a number of other companies have failed in their efforts to develop an effective treatment. In February, Incyte halted trials of its drug Jakafi in solid tumors, after a Phase 3 study in pancreatic cancer failed to show efficacy. And this past May saw pancreatic cancer immunotherapies from NewLink Genetics and Aduro Biotech fall short of goal in Phase 3 and Phase 2b trials, respectively" {September 2016 source}, and

• Momenta Pharmaceuticals has decided to end development of necuparanib, an experimental drug for advanced pancreatic cancer and the biotech's lead clinical candidate, according to a regulatory filing submitted Thursday. Enrollment in a Phase 2 trial testing necuparanib in combination with Celgene's Abraxane was halted earlier this month after an independent data safety monitoring board concluded treatment didn't demonstrate a sufficient level of efficacy {above source}.

Updated (9/28/16): In 2011 co-founder and former Chairman and CEO Dr. Craig Dees, PhD presented Provectus' murine model work examining PV-10's "immunologic potential in treating melanoma and other cancer indications including liver, pancreatic and colorectal cancer." See several pertinent slides below.

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Updated (9/30/16): H/t @bradpalm1: Vitamin D + immunotherapy + chemotherapy..."Physicians and Physicists Join Forces to Fight Pancreatic Cancer," The Wall Street Journal, September 25th, Ron Winslow

"The team is planning a clinical trial soon in which patients who are candidates for pancreatic-cancer surgery first will be treated with synthetic vitamin D, chemotherapy and Bristol-Myers Squibb Co.’s cancer immunotherapy Opdivo. Opdivo is one of the new so-called checkpoint inhibitors that are transforming treatment of cancers such as melanoma and lung cancer. The drugs work by releasing brakes on immune-system T cells, enabling them to pursue tumor cells." 

Bristol-Myers also is combining Opdivo with several other companies and drugs to treat pancreatic cancer, such as Celgene (Abraxane/Paclitaxel protein-bound) and Aduro Biotech (CRS-207 and GVAX)*. Opdivo combination trials for pancreatic cancer on CT.gov include at least eight studies.

* CRS-207 and GVAX failed as a regimen for pancreatic cancer.

Catching On

Updated below.

Takeaways and a Question:

• Getting T cells into cancer tumors is important to more successfully fighting the disease.

• Traditional cancer treatments do not kill enough cancer cells. Immunotherapies like immune checkpoint inhibitors kill more cells than traditional treatments, but they still do not kill enough.

• Ways of getting T cells into tumors include chemotherapy, radiotherapy, targeted therapies and intralesional (IL) or intratumoral (IT) agents like Amgen's talimogene laherparepvec (T-Vec) (Imylgic®) [approved as a single agent], Provectus' PV-10 [in a pivotal Phase 3 trial as a single agent], Viralytics' Cavatak (another oncolytic virus) [having done Phase 2 trial work as a single agent], etc.

• The more (quantity) and better (quality) T cells that get into cancer tumors, the greater the immunologic signalling of the drug or drug compound (immunotherapy) that led to said infiltration.

• If a drug or drug compound's immunologic signalling is so powerful and broad as to enable the Cancer Immunity Cycle to cycle more sustainably and durably, what would have come first, the chicken or the egg (i.e., the left-hand side, or the right-hand side, the antigen cascader or the checkpoint inhibitor)?

Big Pharma-oriented blogger and publisher, and industry consultant, Dr. Sally Church, MD recently wrote a post (on her blog Biotech Strategy Blog; subscription required) asking if chemotherapy was immunotherapy: "Controversy: Is Chemotherapy Immunotherapy?" Her thoughts derived, in part or in whole, from her visit to AACR 2016. She noted in this post (alluding to a prior one) that one of the rate limiting steps in the Cancer Immunity Cycle was "getting more T cells into the tumours so that subsequent immunotherapy can be even more effective." One way to get more T cells into tumors, according to Dr. Church, was chemotherapy.

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Getting T cells into cancer tumors, from the perspective of a traditional treatment like chemotherapy, begins with generating antigens caused by killing cancer cells.

Click to enlarge. Figure 3, Chen and Mellman. Immunity 2013; 39: 1-10.

Utilizing therapeutics or therapies (immunotherapies) from the left-hand side of the Cycle to facilitate the infiltration of presumably educated and trained T cells into cancer tumors, therapeutics (immunotherapies) from the right-hand side can be, to use Dr. Church's words "even more effective."

Following Dr. Church's writing, primarily her tweets (and related or associated material) on Twitter (@maverickNY), is necessary for this investment project. She was kind enough to answer some basic questions of mine early on in my due diligence (basic for her, not so basic for me at the time). With 20 years of experience in the pharmaceutical industry that included the development and launch of Gleevec by Novartis (bios here and here), Dr. Church is smart, intelligent, thoughtful, and (I believe) a weather vane of Big Pharma's market/marketing (and, possibly, R&D) interest and direction. For example, she was and presumably remains a fan of checkpoint inhibitors, but has evolved her views (as I believe them to have developed) in regards to treating late-stage disease from such agents strictly as stand-alone, single agent therapies to their greater relevance and effectiveness in combination with other therapeutics and therapies (presumably stimulatory ones complementary to these inhibitory agents).

AACR 2016 not only discussed chemotherapy as a "potential immunotherapy" (viewing this through Dr. Church's prism), the conference also considered and contemplated radiation therapy or radiotherapy — but fractionated treatment, where radiation treatment is given in several, small doses over a period of time. This is about killing cancer cells to begin the antigen cascade (immunogenic cell death). This is about harnessing the cancer patient's tumor(s) to become a vaccine, so to speak (in situ vaccination).

PV-10 + Chemotherapy. Ironically, as an aside, at SITC 2012 (October), three-and-a-half years before AACR 2016 (April), Provectus presented the results of murine model work combining PV-10 and chemotherapy that generated the best response in non-injected (untreated) tumors {underlined emphasis below is mine}:

"The mechanism by which PV-10's bystander effect is produced was investigated using hepatocellular carcinoma (HCC) and melanoma tumors in immunocompetent and immunodeficient mice. In one set of experiments using immunocompetent mice with bilateral HCC tumors (i.e., two HCC tumors in opposite flanks), intralesional injection of PV-10 into one of the two tumors led not only to eradication of the injected tumor, but also to regression of the uninjected tumor. Controls treated with saline exhibited no effect in either tumor. Treatment of mice with systemic chemotherapy (i.e., 5-fluorouracil, "5-FU") had minimal effect on either tumor, while combination of intralesional PV-10 with systemic 5-FU elicited maximal response in uninjected tumors. Data were analyzed by tumor (i.e., injected tumor, uninjected tumor and total tumor burden) for time to progression and for tumor growth. PV-10 alone was favorable to saline control in all categories, while PV-10 combination therapy produced highly significant advantage vs. control for time to progression of treated tumors (p = 0.010), untreated tumors (p = 0.011) and total tumor burden (p = 0.004)." (Source: Provectus press release, October 2012)

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PV-10 + Radiation. Even more ironic, and as a further aside, Provectus shareholders await the presentation or publication of an investigator-initiated study in Australia combining PV-10 and radiotherapy in patients with advanced melanoma. You know...what the company's CTO Dr. Eric Wachter, PhD said more than a year ago in response to a question of mine.

As background, Radiation therapy has been combined with PV-10 in two situations. First, in 2010, Foote et al. reported on their combination work on 3 patients in A novel treatment for metastatic melanoma with intralesional rose bengal and radiotherapy: a case series, Melanoma Research, 20:48, Jan 2010. All of the patients originally were enrolled in Provectus' metastatic melanoma Phase 2 trial (I believe). All patients achieved complete response (CR) (note detailed descriptions in the article's text); however, no discussion was provided regarding survival (that is, keep in the mind the difference or differentiation between [objective] response rate and overall survival). Note: PV-10 was administered first, followed by radiation.

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Later, in 2013, Tan and Neuhaus reported on their combination work on 2 patients in Novel use of Rose Bengal (PV-10) in two cases of refractory scalp sarcoma, ANZ Journal of Surgery, 83:1-2:93, Jan 2013. Both patients were treated in the company's compassionate use/expanded access program (I believe). One patient achieved a complete response, while the other enjoyed "good" clinical effect before progressing again. Also, no discussion was provided regarding survival of the patient achieving CR. Note: Here, radiation was administered first, followed by PV-10.

During the 4Q15/CY 2014 business update conference call on March 11, 2015, I asked about the timing, and possibly the potential venue(s) (medical conference, journal), of Foote et al.'s clinical follow-up work combining PV-10 and radiotherapy? Eric said: "But regarding the radiation therapy publication by Foote, that is of record as having led to an investigator-initiated study that you mentioned, 25 patients. That study has been enrolling slowly. It had very specific eligibility criteria at a single center. We have had discussions over the last several months with the investigator about ways to get data from that study available publicly and we anticipate that sometime in the coming months, that is sometime this year, that there will be some presentation of interim data from that study" {my underlined emphasis; this year = 2015}.

Step #5, Infiltration of T cells into Tumors. PV-10 has been potentially implicated in each and every step of the Cancer Immunity Cycle, with data on Step #5 to come.

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If PV-10/Rose Bengal can (has) overcome one of the Cancer Immunity Cycle's rate limiting steps (of getting T cells into cancer tumors) in greater quantity and better quality (than other treatments), would Dr. Church then refer to PV-10 as immunotherapy?

Updated (5/20/16): Dr. Church updated or extended her thinking with one of her Blue Ice Publishing episodes at Novel Targets entitled Episode 12: Of Mice and Men. In it she further expands on chemotherapy as immunotherapy, and potentially a partner for other immunomodulatory agents like checkpoint inhibitors.

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Inception to date: PV-10 + [something else]

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